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Detection of cytokeratin 20 mRNA in the peripheral blood of patients with colorectal cancer by immunomagnetic bead enrichment and real‐time reverse transcriptase–polymeras chain reaction
Author(s) -
GUO JUNMING,
XIAO BINGXIU,
JIN ZHIJIN,
QIN LIJUN,
CHEN JIAN,
CHEN HUI,
ZHANG XINJUN,
LIU ZHONG
Publication year - 2005
Publication title -
journal of gastroenterology and hepatology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.214
H-Index - 130
eISSN - 1440-1746
pISSN - 0815-9319
DOI - 10.1111/j.1440-1746.2005.03894.x
Subject(s) - immunomagnetic separation , medicine , colorectal cancer , cytokeratin , peripheral blood mononuclear cell , cancer , circulating tumor cell , ficoll , real time polymerase chain reaction , cancer cell , microbiology and biotechnology , pathology , metastasis , biology , immunohistochemistry , in vitro , gene , biochemistry
Background: Detection of circulating cancer cells is a useful indicator for the risk of recurrence of advanced carcinoma. The aim of the present study was to evaluate the potential value of a novel approach to detect the circulating cancer cells in patients with colorectal cancer. This method is based on a combination of isolation of epithelial cell by a combination of negative and positive immunomagnetic beads with detection of cytokeratin 20 (CK20) mRNA by reverse transcriptase–polymeras chain reaction (RT‐PCR). Methods: Peripheral blood samples were collected from 40 patients with colorectal carcinoma on the day before operation or chemical therapy. Mononuclear cells (MNC) were isolated by centrifugation through a Ficoll gradient. Each MNC sample was equally divided into three parts and then CD45 immunomagnetic beads and/or Ber‐EP4 immunomagnetic beads were used to enrich colon cancer cells. Finally, the CK20 mRNA was detected by real‐time quantitative RT‐PCR. As a control, LS174T colon cancer cells were serially diluted with blood from healthy individuals. Results: When CD45 and Ber‐EP4 immunomagnetic beads were used successively, a significant correlation between CK20 mRNA levels and the initial cell concentrations was found in the control recovery experiment. The sensitivity of the assay was one cancer cell in 1 mL healthy blood. In the patient group, CK20 mRNA was detected in 80.0%, 82.5% and 72.5% of patients when CD45, Ber‐EP4, and CD45/Ber‐EP4 immunomagnetic beads were used, respectively. The positive detection rates of patients with colorectal carcinoma at Dukes A, B, C, and D stage were 0.0% (0/2), 33.3% (3/9), 86.7% (13/15), and 92.9% (13/14), respectively. The CK20 mRNA positive detection rate in peripheral blood was significantly correlated with tumor diameter ( P < 0.01, χ 2 ), lymphatic metastasis ( P < 0.05) and hepatic metastasis ( P < 0.05), but not with the differentiation of tumor cells. Conclusion: The combined use of negative and positive immunomagnetic beads followed by amplification of CK20 mRNA by means of RT‐PCR is a non‐invasive, sensitive, and specific assay for the detection of circulating colonic cancer cells. © 2005 Blackwell Publishing Asia Pty Ltd